We have discovered a family of at least 20 proteins, termed RGSs, that impairs signal transduction through signalling pathways that use seven transmembrane receptors and heterotrimeric G proteins. Such receptors, when activated following the binding of a ligand such as a hormone or a chemokine, trigger Galpha subunits to exchange GTP for GDP; this causes the dissociation of Galpha and Gbetagamma subunits and resultant downstream signaling. RGS proteins bind Galpha subunits and function as GTPase activating proteins, thereby deactivating the Galpha subunit and facillitating the re-association of Galpha with Gbetagamma. We have shown that RGS proteins have some apparent specificity for Galpha subunits, since different RGS proteins preferentially bind certain G( subunits. However, to date we have not discovered RGS proteins that bind Gsalpha. While the RGS proteins bind poorly to GTP or GDP bound Galpha subunits, they bind with high affinity to a transition state analog of Galpha that is present in the GTP hydrolysis reaction. Thus, by stablizing this transition state they likely enhance the intrinsic Galpha GTPase activity. We have created a series of RGS4 mutant proteins, which has revealed several key residues for Galpha binding. Two of the mutant proteins antagonize wild-type RGS activity in GAP and functional assays. We have also found that RGS proteins have different subcellular locations. RGS3 localizes predominantly at the cell membrane as does a C-terminal truncation that lacks an RGS domain. RGS3 also interacts in a phosphorylation dependent way with 14-3-3 proteins, a class of proteins involved in the regulation of signaling molecules. In contrast to RGS3, RGS4 is predominantly cytoplasmic although it can be recruited to the plasma membrane by an activated Galpha subunit. In addition, RGS4 may be recruited to intracellular membranes where it may have a role in regulating secretory processes. Several new RGS family members have been identified and we are characterizing their G( subunit specificity and potency in functional assays. Long-term projects include RGS gene targeting and transgenic expression studies.